The invention relates to a radioimmunological method of determining antigens, in particular hepatitis-B-surface antigen, or antibodies in human body liquids, such as human plasma, human serum and fractions thereof, by forming an antigen/antibody-complex in which the antibody component is radioactively labelled.
Radioimmunotests--also called radioimmunoassays--initially were used for demonstrating the presence of hormones. The system described by Yalow and Berson (J. Clin. Invest. 1157-1175, 1960, "Immunoassay of endogenous plasma insulin in man") is based on the ability of a highly specific antibody to bind its radioactively labelled antigen and on the inhibition of this reaction by the unlabelled (sample) antigen whose presence is to be demonstrated. Thereupon the radioactive antigen which has not been bound is quantitatively removed from the test mixture and the bound activity is measured with a suitable appliance. The difficulty inherent in carrying out such determinations or demonstrations consists in the quantitative separation of the components not reacted in the complex formation. For the separation, the methods of paper electrophoresis, precipitation with a second antibody (double antibody methods), precipitation of the antigen/antibody-complex with polyethyleneglycol, adsorption of the free antigen on activated carbon, or separation by reaction on a solid phase are eligible. In the latter case, the solid phase is produced by a covalent bond of a reaction component on either activated cellulose, Sephadex or Sepharose (Wide and Porath, Biochem. Biophys. Acta 130, 257-260, 1966, "Radioimmunoassay of proteins with the use of Sephadex coupled antibodies"), or by adsorption of a reaction partner to a suitable surface, preferably plastics materials (Catt, Niall and Tregear, Biochem. J., 100, 31C-33C, 1966, "Solid-phase radioimmunoassay of human growth hormone").
For demonstrating hepatitis-B-surface antigen (HB.sub.s AG) and the corresponding antibody (antiHB.sub.s AB) by means of radioimmunoassays, hitherto the following methods have been known:
1. Ausria I, Ausria II, Ausab (Abbott Laboratories). These methods work according to the so-called sandwich-principle. PA1 2. Riausure-method of Electro Nucleonics Laboratories. With this method a sandwich-complex is formed in a similar manner as with the Ausria-II-method, but glass particles are used as solid carriers instead of balls; PA1 3. Combi-RIA-Biotest-method. Here the sample to be examined for antigen is mixed with an adjusted antibody and the mixture is transferred into a small cup that is coated with antigen. After incubation of the sample and washing of the cup, radioactive antigen is added. Then the sample is incubated again and the cup is washed again.
In the Ausria-I-method a tube loaded with antibody is filled with the sample to be examined. After incubation an antibody/antigen-complex (AB-AG) forms, if antigen is present, and the antibody component is fixed to the tube. Then the liquid is removed and the tube is washed and treated with a solution of radioactively labelled antibody. After a repeated incubation, an antibody/antigen/labelled antibody (AB-AG-AB*) complex forms. The tube is washed again and the radioactivity of the AB-AG-AB* complex (sandwich-complex) is determined. The antibody is labelled by installing radioactive iodine (.sup.125 I) in a known manner. The Ausria-II-method uses ball-shaped solid carriers instead of tubes. The Ausab-method is largely similar to the Ausria-methods. Here, however, an inverted set-up of the sandwich-complex is chosen, i.e. AG-AB-AG*;
This is also one of the sandwich-methods, in which complicated washing and separating operations are necessary. If the samples contain only little antigen, all these methods are inaccurate.